PROJECT SUMMARY/ABSTRACT Dramatic increases in insulin resistance (IR) prevalence are expected in the U.S. and throughout the world in coming years. Since IR is an important risk factor for cardiovascular disease, discovery of more efficient ways of preventing and treating this condition would have a huge public health impact. Over the past decades, development of new drugs aimed at preventing cardiometabolic disease has slowed down substantially, but recent advances in human genetics offer new exciting opportunities for drug development. Genome-wide associations studies (GWAS) have discovered >150 loci associated with IR and closely related traits over the past decade; but for the vast majority of these, the causal gene has not been definitely identified and the mechanisms leading to IR are unknown. We have performed colocalization analyses to prioritize 50 plausible candidate genes from 164 GWAS loci associated with IR-related traits, and we now aim to establish and characterize genes causally associated with IR using a rigorous series of experiments combining CRISPR-based gene perturbation with single-cell RNA sequencing and detailed phenotyping in human adipocytes, skeletal myocytes and mouse models. In Aim 1, we will perform CROP-seq ? CRISPR-based transcriptional interference (CRISPRi) followed by single- cell RNA-seq (scRNA-seq) ? in human adipocytes to characterize differentially expressed genes and pathways after knockdown of 50 genes selected based on colocalization analyses. In Aim 2, we will evaluate metabolic phenotypes, such as glucose uptake, lipolysis, insulin signaling, adipogenesis, mitochondrial function, fatty acid oxidation, and metabolite profiles in human adipocytes and skeletal myocytes after CRISPRi knockdown of 25 genes, guided by expression profiles from aim 1. In Aim 3, we will create and breed knockout mouse models for three IR-related genes, and then compare wildtype and knockout mice with regards to fat distribution, glucose and insulin tolerance, energy expenditure, physical activity, food intake, lipid profiles, kidney and liver panels, cellular transcriptome, and histopathology of different tissues in mice on chow and after high-fat feeding. By combining a range of innovative methods including high-throughput gene perturbations followed by single cell transcriptomics, in vitro and in vivo experiments to characterize loci established using human genetics, we expect to establish causal genes and mechanisms of action for novel genes involved in development of IR. This is a first important step towards development of new drugs to address the huge and increasing unmet need posed by IR. Our proposal integrates a range of innovative approaches in different model systems providing a translational framework that is likely to lead to new important insights into insulin resistance, type 2 diabetes and cardiovascular disease which could have a huge public health impact.